Neurulation in the mouse I. The ontogenesis of neural segments and the determination of topographical regions in a central nervous system

Ontogenesis of neural segments and positional relationships between the segments and other organs during neurulation were studied in 1,423 ICR mouse embryos by binocular dissecting, light, and scanning electron microscopy. Late in the presomite stage, two transverse sulci, preotic and otic, were seen on the prospective luminal surface of the neural folds. By somite stage 19, the former subdivided into five neuromeres, and by somite stage 21, the latter subdivided into four neuromeres. From the rostral, preotic sulcus, moreover, five other neuromeres were formed by somite stage 20, and between the otic sulcus and the first somite, two neuromeres were formed by somite stage 28. In the caudal part, from the level of the first somite, a total of 39 neuromeres were formed one after another by somite stage 39, and their positions almost correlated with each corresponding somite. Furthermore, the isthmus grew in the boundary between the fifth and sixth neuromere. The most protruding zone in the preotic sulcus formed the eighth neuromere and was located adjacent to the first branchial arch and the trigeminal ganglion. The most protruding zone in the otic sulcus also formed the 11th neuromere and was located adjacent to the second branchial arch. The 12th and 13th neuromeres were situated adjacent to the otic vesicle; the 23rd to 28th neuromeres, adjacent to the forelimb bud; and the 40th to 46th neuromeres, adjacent to the hindlimb bud.     

Segmentation in staged human embryos: the occipitocervical region revisited

The first seven somites, the rhombomeres, and the pharyngeal arches were reassessed in 145 serially sectioned human embryos of stages 9-23, 22 of which were controlled by precise graphic reconstructions. Segmentation begins in the neuromeres, somites and aortic arches at stage 9. The following new observations are presented. (1) The first somite in the human, unlike that of the chick, is neither reduced in size nor different in structure, and it possesses sclerotome, somitocoel and dermatomyotome. (2) Somites 1-4, unlike those of the chick, are related to rhombomere 8 (rather than 7 and 8) and are caudal to pharyngeal arch 4 (rather than in line with 3 and 4). (3) Occipital segment 4 resembles a developing vertebra more than do segments 1-3. (4) The development of the basioccipital resembles that of the first two cervical vertebrae in that medial and lateral components arise in a manner that differs from that in the rest of the vertebral column. (5) The two groups of somites, occipital 1-4 and cervical 5-7, each form a median skeletal mass. (6) An 'S-shaped head/trunk interface', described for the chick and unjustifiably for the mouse, was not found because it is not compatible with the topographical development of the otic primordium and somite 1, between which neural crest migrates without hindrance in mammals. (7) Occipital segmentation and related features are documented by photomicrographs and graphic interpretations for the first time in the human. It is confirmed that the first somite, unlike that of the chick, is separated from the otic primordium by a distance, although the otic anlage undergoes a relative shift caudally. The important, although frequently neglected, distinction between lateral and medial components is emphasized. Laterally, sclerotomes 3 and 4 delineate the hypoglossal foramen, 4 gives rise to the exoccipital and participates in the occipital condyle, 5 forms the posterior arch of the atlas and 6 provides the neural arch of the axis, which is greater in height than the arches of the other cervical vertebrae. Medially, the perinotochord and migrated sclerotomic cells give rise to the basioccipital as well as to the vertebral centra, including the tripartite column of the axis. Registration between (1) the somites and (2) the occipital and cervical medial segments becomes interrupted by the special development of the axis, the three components of which come to occupy the height of only 2 1/2 segments.     

Second branchial arch lineages of the middle ear of wild-type and Hoxa2 mutant mice.

Our current understanding of the evolution of the mammalian middle ear was first suggested by embryological studies from the 19th century. Here, site-specific recombinase-mediated lineage tracing was used to define the second branchial arch contribution to the middle ear of wild-type and Hoxa-2 mutant embryos. The processus brevis of the malleus was found to arise from second arch tissues, making it the likely homologue of the retroarticular process of nonmammalian tetrapods. The second arch also formed a portion of the otic capsule. In light of avian lineage studies, second arch cells were probably incorporated into the otic capsule before avian and mammalian lineages diverged. In Hoxa2 mutant embryos, middle ear skeletal duplications occurred at sites where first and second arch elements are normally apposed. The dorsoventral positions at which second arch skeletal elements formed and the early migration of second arch neural crest cells were not altered by the absence of Hoxa2 function.     

Expression of mouse Foxi class genes in early craniofacial development.

Recent models of craniofacial development suggest the existence of a common pan-placodal domain lying next to the neural plate, from which all sensory placodes will arise. In support of this idea, several genes are expressed in the surface ectoderm of the head adjacent to the neural plate, before the appearance of genes in specific cranial placodes. In this study, we examine the expression patterns of the mouse Foxi class genes from embryonic day 6.5 to 10.5. Foxi2 is expressed throughout the cranial ectoderm adjacent to the neural plate from the 4-somite stage, later becoming excluded from the otic placode. Foxi3 is expressed in a broad region of the pan-placodal ectoderm adjacent to the neural plate from embryonic day (E) 6.75 to the first somite stage. Its expression becomes restricted to the ectoderm and the endoderm of the branchial pouches at E10.5. Foxi1 expression is first detected in the endolymphatic duct in the otic vesicle at E10.5. These results suggest that the mouse Foxi class genes may play important roles, both during cranial placode specification and in later development of individual cranial sensory structures and other organs derived from the cranial ectoderm.     

Timing of odontogenic neural crest cell migration and tooth-forming capability in mice.

The mammalian tooth develops through sequential and reciprocal interactions between cranial neural crest (CNC)- derived ectomesenchymal cells and the stomadial epithelium. Classic tissue recombination studies demonstrated that premigratory CNC cells and CNC-derived ectomesenchymal cells possess odontogenic capacity and can respond to oral epithelial signals to form a tooth, suggesting that the CNC cells contributing to odontogenic tissue are not prespecified. Here we show that, in mice, CNC cells have populated the forming first branchial arch before the 9-somite stage and continue to migrate into the arch by the 13-somite stage. Grafts of the first arch from the 10-somite embryo or earlier yielded membranous bone and cysts but no teeth after subrenal culture. However, grafts of the first arch with its dorsally adjacent tissue containing migrating neural crest cells from the same age embryos gave rise to teeth. In contrast, teeth formed in first arch grafts that do not contain migrating neural crest cells from embryos with 12 or more somites. Interestingly, the acquisition of tooth forming capability in the first arch coincides with the onset of Fgf8 expression in the oral epithelium. These results suggest that there exists a population of odontogenic neural crest cells that migrates into the first arch between the 10- and 12-somite stages. These cells either possess odontogenic potential and are able to initiate tooth development, or can respond to odontogenic signals derived from the oral epithelium to support tooth formation.     

Embryonic development of the house shrew (Suncus murinus)

Summary The embryonic development during the period from 1 to 12 pairs of somites was observed in an insectivore species, the house shrew (Suncus murinus), which has been bred within a closed colony. Embryos were staged by the number of somite pairs. Each stage was punctuated at every addition of three pairs of somites and numbered after the Carnegie system. The first somite became apparent between 8 and 9.0 days after fertilization, and the 12th somite appeared between 9.5 and 10.0 days. The rate of somite formation was one pair in every 3–4 h on average. The embryonic events during this period were as follows: 1. From the beginning of stage 9, the embryonic body consistently displayed a kyphosis, and as development progressed, the caudal portion of the embryo spiralled clockwise. 2. The first and second pharyngeal arches formed; their development was precocious among mammalian embryos in relation to somitic count. 3. The segmental pattern of the neural fold was similar to that of laboratory rodents and primates. The first fusion of the cranial neural folds took place in the occipital somite region, the second fusion in the diencephalic region, and the third at the end of the neural plate, thus leaving two neuropores in the cephalic region. 4. The timing of appearance of the optic sulcus was similar to that of human embryos but was delayed in comparison with that of laboratory rodents. 5. The heart always showed a more advanced state than that of other mammalian embryos. From the beginning of stage 9, an unpaired endocardial tube was seen in the bulbo-ventricular region, and deflection from a symmetrical appearance soon took place. 6. The differentiation of foregut was also precocious, and the thyroid and respiratory primordia appeared earlier than in other mammals. The present study emphasizes that there are considerable variations in timing and manner of morphogenesis among early mammalian embryos.     

Multilineage differentiation of ectomesenchymal cells isolated from the first branchial arch

Cranial neural crest-derived ectomesenchymal cells may be pluripotent stem cells that are capable of generating a range of phenotypes. The fate of these cells appears to be determined in part by intrinsic genetic programs and also by the influence of extracellular signals in the local environment. The extent of lineage determination once neural crest cells have migrated to the first branchial arch is not clear, although branchial arch pattern is not thought to be the result of crest predetermination. The aim of the present study was to test the hypothesis that ectomesenchymal cells of the first branchial arch show properties of pluripotent stem cells, the lineage of which may be directed by specific molecular signaling. Ectomesenchymal cells were enzymatically isolated from the mandibular processes of BALB/c mice and maintained in an undifferentiated state while cultured with leukemia inhibitory factor or induced to differentiate by lineage-specific induction factors or growth conditions, including transforming growth factor beta, forskolin, and a mineralization-promoting medium. Morphological observations and immunocytochemistry demonstrated that cells could be induced to differentiate into smooth muscle cells, glial cells, and osteoblasts, respectively. In the presence of the mineralization-promoting medium, alkaline phosphatase activity increased significantly and mineralization nodules formed. The data reported support the concept that many, although not all, first branchial arch-derived ectomesenchymal cells show properties of multipotent stem cells, the subsequent fate of which can be influenced by induction factors and growth conditions. Some cells, however, showed a degree of commitment with respect to their fate. The possible application of first branchial arch-derived stem cells to tissue engineering of the orofacial tissues should involve consideration of the developmental stage of cell harvesting and the desired cell fate.     

Role of the neural crest in development of the cartilaginous cranial and visceral skeleton of the medaka, Oryzias latipes (Teleostei)

Summary Neural crestectomies were performed on neurula stage medaka embryos to remove neural crest with tungsten needles from one of five anteriorly located zones. The embryos were allowed to develop to stage 35 (immediately posthatching) larvae, then cleared and stained for cartilage. An analysis of changes to the head skeletons indicated that most of the anterior neurocranium and the entire viscerocranium received neural crest contributions during development. The elements involved included; the lamina orbitonasalis of the nasal capsule, the trabeculae, Meckels' cartilage and the quadrate of the lower jaw, the pterygoid process, the orbital cartilages and the epiphyseals of the neurocranial roof, as well as all the elements of the hyoid and branchial arches. By further analysis of only those neural crest ablations which produced alterations to the head skeleton, the neural crest cells which contributed to the development of each element were mapped. They originated principally, from one of three regions; the mesencephalon (second most anterior zone removed, number II), the preotic rhombencephalon (zone III), or the postotic rhombencephalon (zone IV). Neural crest from the level of the prosencephalon (zone I) was not chondrogenic nor was neural crest from the fifth region (zone V) which extended beyond the 5^th to about the 8^th or 10^th somite and marked the anterior end of trunk neural crest. The data are discussed and are found to be consistent with the results from other vertebrates and support the central role of the neural crest in the development and evolution of the vertebrate bead skeleton.     

Cloning and expression analysis of the chick DAN gene, an antagonist of the BMP family of growth factors.

Differential screening-selected gene aberrative in neuroblastoma (DAN) is a member of a cystine knot protein family that includes Cerberus and Gremlin. First isolated in a screen to identify genes down-regulated in transformed rat fibroblasts, DAN has subsequently been cloned in Xenopus, mouse, and human. Overexpression of DAN suppresses the transformed phenotype and retards the cell's entry into S phase. Biochemical analyses have demonstrated DAN's ability to bind bone morphogenetic proteins and antagonize their signaling activity. In this study, chick DAN was cloned and sequenced, revealing a conserved cystine knot region as well as an N-glycosylation site. A riboprobe was designed from the 3' chick DAN coding sequence and used for analysis of DAN in the developing chick embryo by in situ hybridization. Chick DAN was expressed beginning at stage 10 in the developing somites and the medial otic epithelium. Expression in the neural layer of the eye became apparent at stage 14. By stage 17, expression had expanded to the base of the hindbrain. Limb bud labeling began at stage 20, whereas expression in the branchial arches appeared at stage 25. Chick DAN expression generally corresponded to that of mouse DAN expression as shown by comparative in situ hybridization. However, chick DAN was found in the otic epithelium and notochord, whereas mouse DAN was restricted to the overlying otic ectomesenchyme and was absent from the notochord. This observation suggests that DAN may play different roles in chick and mouse otic and notochord development.     

The development of the human brain, the closure of the caudal neuropore, and the beginning of secondary neurulation at stage 12

Summary Twenty-four embryos of stage 12 (26 days) were studied in detail and graphic reconstructions of five of them were prepared. The characteristic features of this stage are 21–29 pairs of somites, incipient or complete closure of the caudal neuropore, and the appearance of upper limb buds. The caudal neuropore closes during stage 12, generally when 25 somititc pairs are present. The site of final closure is at the level of future somite 31, which corresponds to the second sacral vertebral level. Non-closure of the neuropore may be important in the genesis of spina bifida aperta at low levels. The primitive streak probably persists until the caudal neuropore closes, when it is replaced by the caudal eminence or end-bud (Endwulst oder Rumpfknospe). The caudal eminence, which appears at stage 9, gives rise inter alia to hindgut, notochord, caudal somites, and the neural cord. The material for somites 30–34 (which appear in stage 13) is laid down during stage 12, and its absence would be expected to result in sacral agenesis. Aplasia of the caudal eminence results in cloacal deficiency and various degrees of symmelia.The junction of primary and secondary development (primäre und sekundäre Körperentwicklung) is probably at the site of final closure of the caudal neuropore. Secondary neurulation begins during stage 12. The cavity of the already formed spinal cord extends into the neural cord, and isolated spaces are not found within the neural cord. Primary and secondary neurulation are probably coextensive with primary and secondary development of the body, respectively. The telencephalon medium has enlarged two mesencephalic segments (M1 and M2) are distinguishable, and rhombomere 4 is reduced. The sulcus limitans is detectable in the spinal cord and hindbrain (RhD), and in the mesencephalon and diencephalon, where it extends as far rostrally as the optic sulcus in D1. A marginal layer is appearing in the rhombencephalon and mesencephalon. The first nerve fibres are differentiating, chiefly within the hindbrain (from the nucleus of the lateral longitudinal tract). Optic neural crest is at its maximum, and the otic vesicle is giving crest cells to ganglion 7/8. Neural crest continues to develop in the brain and contributes to cranial ganglia 5, 7/8, and 10/11. The spinal crest extends as far caudally as somites 18–19 but shows no subdivision into ganglia yet. Placodal contribution to the trigeminal ganglion is not certain at stage 12. Such a contribution to ganglion 7/8 is not unlikely. Involvement of neural crest in the formation of the derivatives of pharyngeal arches 1 and 2 is possible but has not yet been confirmed in the human embryo.Supported by research grant No. HD-16702, Institute of Child Health and Human Development, National Institutes of Health (USA)     

Development of the embryonic chick otic placode. I. Light microscopic analysis

At the 7–8 somite stage of embryonic chick development (29-31 hours of incubation), a slightly elliptical island of thickened ectoderm appears laterally on either side of the most distal constricture of the rhombencephalon at the level of the anterior intestinal portal. The appearance and extent of this auditory placode is precisely correlated with the subjacent accumulation of neural crest cells. By 33 hours of incubation, there is a distinct depression in the developing otic placode, and by 40 to 45 hours, the placode is visibly invaginated, forming an epithelial vesicle or otocyst. Carefully staged embryos were serially sectioned, and the area underlying the developing otic placode was traced with a planimeter. It was found that placode size (area 60,000 μm²) is nearly unchanged from 30 to 42 hours of development. During this time interval, the placode cells first become columnar, show nuclear orientation, and then pseudostratify. The increase in placode cell number during this time interval is not likely to be the result of localized, accelerated cell division: the population doubling time of placode cells is eight and one-half hours and the mitotic index of 2.5% is similar to that of cells in an equivalent area of adjacent, non-placode forming head ectoderm. A model of otic placode formation is proposed which suggests that by 30 hours of development, a discrete population of placode forming cells is segregated from head ectoderm. Subsequent epithelial pseudostratification results from accumulation of this dividing population within the limits of the placode.     

The development of the human brain and the closure of the rostral neuropore at stage 11

Summary Twenty embryos of stage 11 (24 days) were studied in detail and graphic reconstructions of twelve of them were prepared. The characteristic feature of this stage is 13–20 pairs of somites.The notochord sensu stricto appears first during this stage, and its rostral and caudal parts differ in origin. Rostrally, the notochordal plate is being transformed into the notochord in a caudorostral direction. The caudal part, however, arises from the axial condensation in the caudal eminence in a rostrocaudal direction. The caudal eminence (or end bud) represents the former primitive streak. The somites are increasing in number at a mean rate of 6.6 h per pair.The rostral neuropore closes towards the end of stage 11. The closure is basically bidirectional, being more rapid in the roof region and producing the embryonic lamina terminalis and future commissural plate in the basal region. The caudal neuropore is constantly open. The brain comprises telencephalon medium (represented by the embryonic lamina terminalis) and a series of neuromeres: 2 for the forebrain (D1 and D2), 1 for the midbrain, and 6–7 for the hindbrain (RhA-C; Rh D is not clearly delineated). The forebrain still occupies a small proportion of the total brain, whereas the spinal part of the neural tube is lengthening rapidly. Some occlusion of the lumen of the neural tube was noted in 4 embryos, all of which had an open rostral neuropore. Hence there is at present no evidence that occlusion plays a role in expansion of the human brain. The marginal (primordial plexiform) layer is appearing, particularly in rhombomere D and in the spinal portion of the neural tube. The neural crest is still forming from both the (open) neural groove and the (closed) neural tube, and exclusively from both neural (including optic) and (mainly) otic ectoderm.The optic sulcus is now prominent, and its wall becomes transformed into the optic vesicle towards the end of stage 11. At this time also, an optic sheath derived from mesencephalic crest and optic crest is present. The mitotic figures of the optic neural crest are exceptional in being situated in the external part of the neural epithelium. The otic pit is becoming deeper, and its wall is giving rise to neural crest that is partly added to the faciovestibulocochlear ganglion and partly forms an otic sheath. The nasal plate does not yet give off neural crest.Abbreviations: Figs. 1–10. a Endoderm caudal to neurenteric canal or its site B Endoderm rostral to neurenteric canal or its site - A-H Primordium of adenohypophysis - All Allantoic primordium - Ao Aorta - C.E. Caudal eminence (caudal bud, end bud) - Caud.lim.S. Caudal limiting sulcus - Ch. Chiasmatic plate - D Diencephalon - F Foregut, pharynx - Cl. Cloacal membrane - Ggl Ganglion - H Hindgut - I Infundibulum - L.T. embryonic lamina terminalis - M Mamillary area - Mes. Mesencephalon, mesencephalic - Mit. Mitotic figure - Nas. Nasal plate - N.C. Site of neurenteric canal - N.Cr. Neural crest - Not.Pl. Notochordal plate - Not. Notochord - O-Ph Oropharyngeal membrane - Opt. Optic primordium - Opt.S. Optic sulcus - Ot. Otic pit - Ot.sh. Otic sheath - Ph.Ar. Pharyngeal arch - Pr. Mesenchyme of prechordal plate - Pros. Prosencephalon - Rec. Postoptic recess - Resp. Respiratory primordium - Rh Rhombomere - S.V. Sinus venosus - s. Somite - Tel. Telencephalon - Th. Chickening - Thyr. Thyroid primordium - Trig. Trigeminal - X Cau - al neuropore - Y Rostral neuropore Supported by research grant No. HD-16702, Institute of Child Health and Human Development, National Institutes of Health (USA)     

Fgfr1 regulates patterning of the pharyngeal region

Development of the pharyngeal region depends on the interaction and integration of different cell populations, including surface ectoderm, foregut endoderm, paraxial mesoderm, and neural crest. Mice homozygous for a hypomorphic allele of Fgfr1 have craniofacial defects, some of which appeared to result from a failure in the early development of the second branchial arch. A stream of neural crest cells was found to originate from the rhombomere 4 region and migrate toward the second branchial arch in the mutants. Neural crest cells mostly failed to enter the second arch, however, but accumulated in a region proximal to it. Both rescue of the hypomorphic Fgfr1 allele and inactivation of a conditional Fgfr1 allele specifically in neural crest cells indicated that Fgfr1 regulates the entry of neural crest cells into the second branchial arch non-cell-autonomously. Gene expression in the pharyngeal ectoderm overlying the developing second branchial arch was affected in the hypomorphic Fgfr1 mutants at a stage prior to neural crest entry. Our results indicate that Fgfr1 patterns the pharyngeal region to create a permissive environment for neural crest cell migration.     

The development of the neural crest in the human

The first systematic account of the neural crest in the human has been prepared after an investigation of 185 serially sectioned staged embryos, aided by graphic reconstructions. As many as fourteen named topographical subdivisions of the crest were identified and eight of them give origin to ganglia (Table 2). Significant findings in the human include the following. (1) An indication of mesencephalic neural crest is discernible already at stage 9, and trigeminal, facial, and postotic components can be detected at stage 10. (2) Crest was not observed at the level of diencephalon 2. Although pre-otic crest from the neural folds is at first continuous (stage 10), crest-free zones are soon observable (stage 11) in Rh.1, 3, and 5. (3) Emigration of cranial neural crest from the neural folds at the neurosomatic junction begins before closure of the rostral neuropore, and later crest cells do not accumulate above the neural tube. (4) The trigeminal, facial, glossopharyngeal and vagal ganglia, which develop from crest that emigrates before the neural folds have fused, continue to receive contributions from the roof plate of the neural tube after fusion of the folds. (5) The nasal crest and the terminalis-vomeronasal complex are the last components of the cranial crest to appear (at stage 13) and they persist longer. (6) The optic, mesencephalic, isthmic, accessory, and hypoglossal crest do not form ganglia. Cervical ganglion 1 is separated early from the neural crest and is not a Froriep ganglion. (7) The cranial ganglia derived from neural crest show a specific relationship to individual neuromeres, and rhombomeres are better landmarks than the otic primordium, which descends during stages 9-14. (8) Epipharyngeal placodes of the pharyngeal arches contribute to cranial ganglia, although that of arch 1 is not typical. (9) The neural crest from rhombomeres 6 and 7 that migrates to pharyngeal arch 3 and from there rostrad to the truncus arteriosus at stage 12 is identified here, for the first time in the human, as the cardiac crest. (10) The hypoglossal crest provides cells that accompany those of myotomes 1-4 and form the hypoglossal cell cord at stages 13 and 14. (11) The occipital crest, which is related to somites 1-4 in the human, differs from the spinal mainly in that it does not develop ganglia. (12) The occipital and spinal portions of the crest migrate dorsoventrad and appear to traverse the sclerotomes before the differentiation into loose and dense zones in the latter. (13) Embryonic examples of synophthalmia and anencephaly are cited to emphasize the role of the neural crest in the development of cranial ganglia and the skull.     

Molecular differentiation of the otic vesicle and neural tube in the chick embryo demonstrated by monoclonal antibodies

Monoclonal antibodies (MAbs) were produced against membrane fractions of the chick neural tube and somite. These MAbs selectively stained the neural tube and neural crest cells; the antigens for some of these MAbs were identified as cell adhesion molecules or glycolipids. Histochemistry of the otic vesicle and its progeny in the chick embryo was performed with these MAbs and other MAbs obtained previously in our laboratory. It was demonstrated that differentiation of the otic placode and vesicle from the ectoderm, and development of the acoustic ganglion from the otic vesicle were accompanied by the appearance and disappearance of various molecules.     

The myth of ventrally emigrating neural tube (VENT) cells and their contribution to the developing cardiovascular system

The cardiac neural crest cells are a group of cells that emigrate from the dorsal side of the neural tube during a specific time window and contribute to the pharyngeal arch arteries and the aorticopulmonary septum of the heart. Recent publications have suggested that another group of cells emigrating from the ventral side of the neural tube also contributes to the developing cardiovascular system. The first aim of our study was to define the specific time window of cardiac neural crest cell migration by injecting a retrovirus containing a lacZ reporter gene into a chick embryo at different stages during development. The second aim was to study the contribution of the supposed ventrally emigrating neural tube cells to the cardiovascular system using three approaches. One approach was to inject a lacZ retrovirus into the lumen of the chick hindbrain. Secondly, we injected the retrovirus into the neural tube at the position of the 10-12 somite pair. Finally, we used the chimera technique in which we transplanted a quail neural tube segment into a chick embryo. Cardiac neural crest cells were shown to emigrate from the dorsal side of the neural tube between HH9 and HH13(-). The HH13(+) neural tube has ceased to produce cardiac neural crest cells between the level of the otic placode and the fourth pair of somites. Retroviral injection directly into the chick hindbrain at HH14 resulted in 50% of the embryos with minimal labeling of the hindbrain and intense labeling of the adjacent mesenchyme, suggesting that virus was spilled. This implies that this technique is not useful for confirming the existence of ventrally emigrating cells. Both retroviral injections into the neural tube lumen at HH14 at the position of the 10-12 somite pair and the chimeras showed no signs of ventrally emigrating neural tube cells. We conclude that there is no contribution of ventral neural tube cells to the developing cardiovascular system.